The purified capsule of H. influenzae type b (Hib) is a poor immunogen in human infants, the age group a greatest risk of developing Hib disease. Outer membrane proteins (OMP) are immunogenic in infant animals and human infants recovered from Hib disease. Antisera directed against OMPs are protective against experimental Hib bacteremia in infant rats. We have purified and partially characterized the major OMPs of Hib. The most abundant OMP, P2, binds lipopolysaccharide and has porin activity. Antibody directed against OMP P2 protects against experimental bacteremia in infant rats challenged with the strain from which we prepared the immunogen. This protection is unaffected by absorption of anti-LPS antibody. OMP P2 is present in all Hib strains examined. Preliminary peptide analysis indicates that these proteins are conserved proteins although immunofluorescence and rat protection data indicate heterogeneity of the surface exposed epitopes. We propose to map the surface exposed epitopes of this protein by a combination of chemical, immunologic and genetic approaches. Antisera will be prepared against purified proteolytic and CNBr peptides and tested for protection in the rat model. Monclonal antibodies will be prepared and used to map surface epitopes and to determine their presence in different disease producing isolates. The primary structure of the protein will be determined by DNA sequencing the cloned P2 gene. Through these approaches we will obtain data to construct an antigenic model of the protein. Peptides representative of the surface exposed epitopes will then be synthesized. These peptides will be tested for reactivity with polyclonal and monoclonal antibodies reactive against the native protein. We also will conjugate the synthetic peptides to carrier proteins, immunize animals and assay the antisera for reactivity with the native protein; and determine the protective activity in the infant rat model. In other systems, cross reactive but normally immunorecessive epitopes have been identified by exploiting these methodologies. Conceivably, we can identify broadly crossreactive antigenic determinants that can be synthesized and incorporated into nontoxic vaccines. However, even if immunity is strain-specific, it should be possible to incorporate peptides from representative strains into future vaccines as a limited number of strains produce greater than 90% of Hib disease in this country. The experiments proposed will provide molecular characterization of a major OMP from an important pathogen, and increase our understanding of the use of defined peptides in vaccines.